Project Abstract Adolescence and early adulthood is a period of significant brain development, when different experiences shape the synaptic connections between neurons and information processing is increasingly integrated across various brain regions. It is also when substance use is at its greatest, on average. Research with animals indicates that the brain is particularly vulnerable to damaging effects of alcohol and other drugs during adolescence. However, most individuals moderate their drinking and it is arguably as important to understand the degree to which brain structural and functional effects of alcohol and other drug use tend to be reversed when individuals moderate their use as to understand effects of exposure to alcohol in the first place. However, several factors have slowed progress in this area. Samples have been small, especially in early studies, and the number of potential measures large, which, if properly controlled for, limits statistical power and, if not, increases the likelihood of chance findings. The lack of specific replicated associations suggests a need for studies that use discovery-based methods in large samples in addition to those that test specific hypotheses, and to replicate findings in independent samples. An additional obstacle to progress stems from the difficulty drawing inferences about causality in observational research. Associations between substance use or moderation and brain outcomes may reflect a causal effect of substance use or moderation or they may be spurious, reflecting the effect of a third, unobserved factor, such as a genetic liability, on both. For instance, individuals who stop abusing alcohol or other drugs may have less of a genetic liability toward excessive use than those who persist in problematic drinking, and any observed differences between the two groups of subjects may reflect this difference rather than any result of having desisted from problematic use. The present data analysis project is designed to address these challenges. We propose to study effects of cumulative alcohol use, the timing of exposure to alcohol and effects of moderating use on measures of brain structure and function in three large independent samples, which allows us to replicate findings and thereby increase confidence in them. We will use a combination of hypothesis-driven and discovery-based analyses. In addition, because two samples consist of twins, we will use an innovative co-twin differences research design, which takes advantage of differences in overall alcohol use, age of initiation and moderation of use that naturally occur within twin pairs to separate the effects of patterns of use and moderation from the genetic and other factors that influence them. This will permit us to make stronger inferences than is typically possible about which effects of alcohol use and moderation of use on measures of brain structure and function are likely to indicate a truly causal influence of exposure to alcohol and of reducing one?s exposure, which can inform prevention and intervention efforts as well as ongoing longitudinal studies.